cDNA Library Construction Kits

Create Long-Insert Libraries

The SMART cDNA Library Construction Kit is designed for the cloning of full-length cDNA into a phage TriplEx2 vector. The kit combines the SMART technology (Switching Mechanism At 5' end of RNA Template) for cDNA amplification with adaptor-free, directional cloning into the TriplEx2 vector. This kit contains two separate protocols, allowing you to choose a method based on your starting material. The first protocol employs long-distance PCR (LD PCR), for researchers limited by their starting material. As little as 50 ng of total RNA can be used as starting material (1). The second protocol provides a more straightforward protocol for researchers with abundant amounts of starting material (i.e., 1 µg or more of poly A+ RNA). SMART libraries contain a higher percentage of full-length clones than libraries constructed by conventional methods or other full-length cDNA synthesis protocols. Thus, clones isolated from SMART cDNA libraries contain sequences corresponding to the complete 5' untranslated region of the mRNA (2).

Enrich for Full-Length cDNA

SMART cDNA Synthesis Technology ensures uninterrupted cDNA synthesis, creating cDNAs with well-represented 5’ end sequences. Since terminal transferase activity and the subsequent SMART switching process occur preferentially at the 5' ends of eukaryotic mRNAs, truncated products resulting from premature termination of the reverse transcription reaction generally do not incorporate the SMART(er) oligonucleotide, and consequently are not amplified during PCR. Thus, cDNA pools created using our SMART technology and amplified by long-distance PCR are enriched for full-length cDNA. Additionally, because the 5’ SMART(er) sequence and modified oligo dT primer are not added onto genomic DNA or cDNA transcribed from ribosomal RNA, cDNA that is generated using SMART is free of these contaminating agents.

Save Time with a Streamlined Protocol

The In-Fusion SMARTer Library Construction protocol can be completed in fewer steps than other library construction methods due to a highly efficient cDNA synthesis and cloning process. Only three enzymes are required to complete the entire protocol, as opposed to the usual 6–8 enzymes required for other methods. The SMART(er) cDNA synthesis protocol is user friendly and straightforward with no adaptor ligation or tailing steps. Your precious RNA is subjected to the to the least possible handling, thereby minimizing the risk of degradation.

Insert Your Library Into any Vector

In-Fusion Cloning makes it easy to clone your SMARTer cDNA library into the pSMART2IF or pSMART2IFD linearized vectors (included in In-Fusion SMARTer kits) in just one 30 min reaction. Most importantly, since In-Fusion Cloning is designed to join fragments of DNA with 15 complementary bp at their ends, In-Fusion kits can be used to precisely transfer your SMARTer cDNA into ANY linearized vector. If you would like to clone your library into your own expression vector for functional analysis, simply amplify your vector by inverse PCR using primers that create linear vector ends that are complementary to the ends of the SMARTer cDNA. Primers must have two characteristics: the 5’ end of the primer must contain 15 bases that are complementary to 15 bases at one end of the DNA fragment to which the vector will be joined (i.e., the insert), and the 3’ end of the primer must contain sequence that is specific to the target vector.

Applications

References

Zhu, Y. et al. (July 1996) Clontechniques XI(3):12–13.

Wellenreuther, R. et al. (October 2005) Clontechniques XX(2):24–25.

Additional Information

Please see the product's Certificate of Analysis for information about storage conditions, product components, and technical specifications. Please see the Kit Components List to determine kit components. Certificates of Analysis and Kit Components Lists are located under the Documents tab.